Establishing Debaryomyces hansenii as a superior cell factory for the green transition: optimizing the use of industrial side-streams and complex feedstock for biotech applications

Mònica Estrada Duran*

*Corresponding author for this work

Research output: Book/ReportPh.D. thesis

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Abstract

Switching from a linear to a circular economy is crucial to meet society’s growing demand for goods and services while respecting the environmental limits of our planet. In addition, replacing finite fossil-fuel resources with renewable biological raw materials is essential for fostering more sustainable processes and mitigating climate change. The biotechnological industry plays a key role in this process, as some microorganisms are able to use waste streams as feedstock to grow and produce a wide range of bioproducts, including food, chemicals, drugs, fuels, and energy. In this way, waste can become a raw material for alternative production processes. However, it is necessary to identify new robust cell platforms that can thrive in such harsh and complex environmental conditions and achieve optimal production yields.

Debaryomyces hansenii (D. hansenii) is a halophilic, xerotolerant, and oleaginous non-conventional yeast with a huge potential for this purpose due to its inherent characteristics. It can tolerate high concentrations of salt (up to 4 M NaCl), high osmotic pressures, extreme temperatures and pH levels, metabolize a wide range of carbon sources, and grow with the presence of some fermentation inhibitors, such as furfural, vanillin, or organic acids. These beneficial features render this yeast a well-suited option for revalorizing complex by-products, especially if they are rich in salt.

Hence, the first part of this thesis assesses the potential of D. hansenii to revalorize three different salt-rich industrial by-products from the dairy and pharmaceutical industry to produce recombinant proteins. The yeast was able to grow in the by-products and successfully produce Yellow Fluorescent Protein (YFP, used as a model) without requiring any nutritional supplement or freshwater. Interestingly, open (non-sterile) cultivations at different laboratory scales (1.5 mL, 500 mL, and 1 L) were accomplished due to the high salt concentration of the by-products, which favored D. hansenii’s metabolism and hindered other non-halotolerant microorganisms present in the by-products.

Recently, an efficient CRISPRCUG/Cas9 toolbox to engineer D. hansenii has been developed. However, to streamline the generation of transformant strains for high-throughput screenings, the second part of this thesis demonstrates the feasibility of performing in vivo DNA assembly in D. hansenii. Up to three different DNA fragments containing 30-bp homologous overlapping overhangs were co-transformed into the yeast and fused in the correct order in a single step. This technique was used to screen potential promoters, terminators, and signal peptides to enhance D. hansenii’s production of recombinant proteins in the salty-rich by-products. The highest production of YFP was achieved using the TEF1 promoter (from Arxula adeninivorans) and the CYC1 terminator (from Saccharomyces cerevisiae). Remarkably, D. hansenii was able to secrete YFP using the α-mating factor (MF) signal peptide (from Saccharomyces cerevisiae). The protein remained stable in the supernatant for 140 h despite the elevated salt concentration (1 M NaCl) and osmolarity of the by-products.

Finally, the last part of this thesis investigates D. hansenii’s potential to use Volatile Fatty Acids (VFAs) as a sole carbon source to grow and produce lipids, avoiding the use of expensive sugars (glucose). VFAs can be sustainably produced from lignocellulosic waste via a shortened anaerobic digestion process, eliminating the dependence on fossil fuel resources. D. hansenii was able to grow with up to 15 g/L of total VFAs and metabolize all of them. Notably, higher biomass yields were obtained using a VFAs-rich organic digestate as feedstock compared to synthetic media. In addition, the yeast accumulated up to 20.87% w/w lipids under specific conditions, highlighting its potential for microbial oil production from VFAs-rich sources.

Overall, this thesis aims to exploit the potential of D. hansenii as a host to produce relevant bioproducts from various waste streams (dairy, pharmaceutical industry, and lignocellulosic biomass), which is a promising step towards establishing a circular and bio-based economy. We expect that with all the knowledge presented in this thesis and the ongoing advancements in genetic engineering tools for D. hansenii, we will be able to develop even more efficient cell factories and support the transition to a more sustainable future.
Original languageEnglish
Place of PublicationKgs. Lyngby, Denmark
PublisherDTU Bioengineering
Number of pages227
Publication statusPublished - 2024

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